The purpose of this project is to develop kinetic tools for studying enzyme mechanisms, and to apply them to representative enzymes. During the coming grant period the emphasis will be on the use of 13C, 15N, and 180 isotope effects: l) Isotope effects in the beta- gamma bridge and gamma-nonbridge oxygens of ATP will be used to study the reactions of myosin ATPase and several kinases. 18o isotope effects with several ribonuclease substrates will be used to determine whether the reaction is concerted, or involves a phosphorane intermediate. 18o isotope effects on the hydrolysis of triesters with leaving group pK's from 10-14 will determine whether such reactions involve phosphorane intermediates. Triesters containing a cyclic ethylene group and a leaving group with px 8.6- 14 will permit observation of isotope effects that accompany phosphorane formation and pseudorotation during hydrolysis. 2) With malic enzyme, 13C isotope effects will be measured at C-3 of malate or 2-d-malate with NADP, or with nucleotides where hydride transfer is more rate limiting than decarboxylation to answer the question of whether the mechanism is stepwise or concerted with nucleotides other than NADP. 3) 13C and 15N isotope effects will be measured at C-2 of aspartate and 2-d-aspartate to determine the relative rates of steps in the chemical reaction for wild type aspartate transaminase and mutants in which one of the chemical steps has been slowed down. 4) 13C isotope effects will be determined-at C-3 and C-4 to see if the chemical -mechanism of L-ribulose-5-P 4- epimerase involves a retro aldol cleavage between these carbons, followed by rotation of the aldehyde group and recondensation to the epimerized product. 5) Conditions for reproducible observation of the 31P NMR signal at -5 ppm that may represent carboxy-P in solutions of phosphate in DMF containing half CO2 will be worked out, and the compound tested as a substrate for appropriate enzymes.